-
Heregulin Targets ;-Catenin to the Nucleolusby a Mechanism
Dependent on theDF3/MUC1 Oncoprotein
Yongqing Li,1,2 Wei-hsuan Yu,1 Jian Ren,1 Wen Chen,1 Lei
Huang,1
Surender Kharbanda,1,2 Massimo Loda,1 and Donald Kufe1
1Dana-Farber Cancer Institute, Harvard Medical School and 2ILEX
Products, Inc., Boston, MA
AbstractThe DF3/MUC1 transmembrane oncoprotein is
aberrantly overexpressed in most human breast
carcinomas and interacts with the Wnt effector
;-catenin. Here, we demonstrate that MUC1 associates
constitutively with ErbB2 in human breast cancer cells
and that treatment with heregulin/neuregulin-1 (HRG)
increases the formation of MUC1-ErbB2 complexes.
The importance of the MUC1-ErbB2 interaction is
supported by the demonstration that HRG induces
binding of MUC1 and ;-catenin and targeting of the
MUC1-;-catenin complex to the nucleolus. Significantly,
nucleolar localization of ;-catenin in response to HRG is
dependent on MUC1 expression. Moreover, mutation of a
RRK motif in the MUC1 cytoplasmic domain abrogates
HRG-induced nucleolar localization of MUC1 and
;-catenin. In concert with these results, we show
nucleolar localization of MUC1 and ;-catenin in human
breast carcinomas but not in normal mammary ductal
epithelium. These findings demonstrate that MUC1
functions in cross talk between ErbB2 and Wnt pathways
by acting as a shuttle for HRG-induced nucleolar
targeting of ;-catenin.
IntroductionThe ErbB family of receptor tyrosine kinases
includes
ErbB1/epidermal growth factor receptor (EGFR), ErbB2/neu,
ErbB3, and ErbB4. Activation of ErbB1, ErbB3, and ErbB4 is
conferred by direct binding of at least 10 different growth
factors that induce receptor homodimerization and hetero-
dimerization (1). The ErbB2 receptor, which has no known
ligand, is transactivated through heterodimerization with
the
other ErbB family members (2, 3). Stimulation of EGFR with
the epidermal growth factor (EGF) induces the formation of
EGFR-ErbB2 heterodimers (4). Similarly, heregulin/neuregu-
lin-1 (HRG) binds to the ErbB3 and ErbB4 receptors and
activates ErbB2 through heterodimerization and transphos-
phorylation (5). ErbB2 may thus function as a coreceptor
that potentiates signaling of the other ErbB family members
(6–8). Dimerization of the ErbB receptors results in activa-
tion of the intrinsic kinase function and phosphorylation of
tyrosine residues that serve as binding sites for proteins
that
contain Src homology 2 or phosphotyrosine binding domains
(9, 10). Activation of ErbB2 is also associated with disrup-
tion of epithelial cell polarity and initiation of
proliferation
(11, 12). In normal polarized glandular epithelial cells,
effectors of the Wnt signaling pathway, h- and g-catenin,are
localized to the adherens junction where they function
with E-cadherin in cell-cell interactions (13). Loss of
polarity
as found with ErbB2 activation (11), however, is associated
with catenin translocation from the adherens junction to the
cytoplasm and nucleus (14). A functional relationship
between
ErbB2 signaling and Wnt regulation of catenins is unknown,
although both ErbB2 and Wnt have been linked to the
development of breast carcinomas.
Human DF3/MUC1 is a mucin-like transmembrane glyco-
protein, which is overexpressed by breast and other carcino-
mas (15). MUC1 expression is restricted to the apical
borders
of normal secretory epithelial cells and is aberrantly
expressed
by breast carcinoma cells at high levels over the entire
cell surface (15). Importantly, overexpression of MUC1 is
sufficient to induce transformation (16). The MUC1 protein
consists of a NH2-terminal (N-ter) ectodomain with variable
numbers of conserved 20-amino acid tandem repeats that are
modified by O-glycosylation (17, 18). The f25-kd COOH-terminal
(C-ter) subunit includes a transmembrane domain and
a 72-amino acid cytoplasmic domain (CD). The extracellular
>250-kd ectodomain associates with the C-ter subunit as a
heterodimer. A SAGNGGSSL motif in the MUC1-CD
functions as a binding site for h-catenin (19). The SAGNG-GSSL
motif also serves as a binding site for g-catenin(plakoglobin)
(19). Glycogen synthase kinase 3h (GSK3h)phosphorylates MUC1 on
serine in a SPY site adjacent to that
for h/g-catenin binding and decreases the interaction
betweenMUC1 and h-catenin (20). Conversely, EGFR- or c-Src-mediated
phosphorylation of MUC1 on tyrosine in the SPY
site up-regulates the formation of MUC1-h-catenin complexes(21,
22). The demonstration that MUC1 and E-cadherin, a
transmembrane protein that functions in Ca2+-dependent
epithelial cell-cell interactions (23), compete for binding
to
h-catenin (20) has supported a role for MUC1 in regulating
Received 3/3/03; revised 6/20/03; accepted 6/24/03.The costs of
publication of this article were defrayed in part by the payment
ofpage charges. This article must therefore be hereby marked
advertisement inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.Grant support: National Cancer Institute grant
CA97098. Note: Y.L. and W.-h.Y.contributed equally to this
work.Requests for reprints: Donald Kufe, Dana-Farber Cancer
Institute, HarvardMedical School, Boston, MA 02115. Phone: (617)
632-3141; Fax: (617) 632-2934.E-mail:
[email protected] D 2003 American Association
for Cancer Research.
Vol. 1, 765–775, August 2003 Molecular Cancer Research 765
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
adherens junction function. Other studies have demonstrated
that MUC1 also colocalizes with h-catenin in the nucleus(16,
24). Less is known about the regulation of binding
between MUC1 and g-catenin.The present studies demonstrate that
MUC1 interacts
with ErbB2 and that HRG stimulation of human breast
carcinoma cells is associated with increased binding of
MUC1 and g-catenin. The functional significance of thissignaling
pathway is supported by the finding that HRG targets
g-catenin to the nucleolus by a MUC1-dependent mechanismand that
a RRK motif in MUC1-CD is required for this
response.
ResultsHRG Induces the Association of MUC1 and ErbB2
Previous studies have demonstrated that human ZR-75-1
breast cancer cells express MUC1 and the four ErbB family
members (EGFR and ErbB2–4) (20, 22, 25). To determine
whether MUC1 associates with ErbB2, anti-MUC1 (DF3)
N-ter immunoprecipitates from lysates of human ZR-75-1
cells were analyzed by immunoblotting with anti-ErbB2. The
results demonstrate that ErbB2 coprecipitates with MUC1
(Fig. 1A). Whereas HRG stimulates ErbB2 activity, lysates
were prepared from ZR-75-1 cells treated with HRG for
5 min. Immunoblot analysis of anti-MUC1 immunoprecipi-
tates with anti-ErbB2 demonstrated that HRG stimulates the
formation of complexes containing MUC1 and ErbB2
(Fig. 1A). In the reciprocal experiment, immunoblot analysis
of anti-ErbB2 immunoprecipitates with anti-MUC1 confirmed
that HRG increases the basal association of MUC1 and
ErbB2 (Fig. 1A). Treatment of ZR-75-1 cells with EGF had
little (if any) effect on binding of MUC1 and EGFR (22). As
a control and in contrast to the effects of HRG, treatment
with EGF also had no apparent effect on binding of MUC1
and ErbB2 (data not shown). HRG binds to ErbB3 and ErbB4
and induces their heterodimerization with ErbB2 (3). To
determine whether MUC1 associates with ErbB3 or ErbB4,
immunoprecipitates prepared with antibodies against these
receptors were subjected to immunoblotting with anti-MUC1.
The results show that MUC1 associates with ErbB3 and
ErbB4 (Fig. 1B). Moreover, HRG stimulated the association
of MUC1 with ErbB3 and ErbB4, but to a much lesser extent
than that found for MUC1 and ErbB2 (Fig. 1B). To define
the subcellular localization of MUC1 and ErbB2, confocal
microscopy was performed with mouse anti-MUC1 and rabbit
anti-ErbB2. In control ZR-75-1 cells, MUC1 was distributed
uniformly over the cell membrane (Fig. 1C, left). A similar
pattern was obtained for the distribution of ErbB2 (Fig. 1C,
second panel). Overlay of the signals supported some
colocalization (red + green ! yellow) (Fig. 1C, right).Following
HRG stimulation for 5 min, MUC1 was clustered
in patches on the cell surface (Fig. 1D, left). Staining for
ErbB2 revealed a similar pattern (Fig. 1D, second panel),
and
overlay of the signals showed increased colocalization of
MUC1 and ErbB2 in clusters at the cell membrane (Fig. 1D,
right). There was no apparent HRG-induced localization of
MUC1 N-ter to the nucleus (Fig. 1D). Moreover, as a control,
there was no increased colocalization of MUC1 and ErbB2
in cells stimulated with EGF (Fig. 1E). These findings
demonstrate that colocalization of MUC1 and ErbB2 at the
cell membrane is regulated by HRG stimulation.
HRG Regulates Interaction of MUC1 and c-CateninTo determine
whether HRG affects the interaction be-
tween MUC1 and catenins, lysates from control and HRG-
treated ZR-75-1 cells were subjected to immunoprecipitation
with anti-MUC1. Immunoblot analysis of the precipitates with
anti-h-catenin demonstrated that HRG has little effect onbinding
of MUC1 and h-catenin (Fig. 2A). By contrast, HRGtreatment was
associated with an increase in binding of MUC1
and g-catenin (Fig. 2A). For comparison, ZR-75-1 cells
werestimulated with EGF. As shown previously, EGF induced
binding of MUC1 and h-catenin (22) (Fig. 2B). Conversely,EGF had
little effect on the interaction of MUC1 with
g-catenin (Fig. 2B). To extend these findings, we used
humanHCT116 carcinoma cells that are MUC1 negative as
determined by immunoblotting with anti-MUC1 antibodies
and by reverse transcription-PCR for sequences encoding the
C-ter [(26) and data not shown]. Moreover, flow cytometric
analysis of HCT116 cells demonstrated that all four ErbB
family members are expressed at the cell membrane and that
ErbB2 is detectable at somewhat higher levels than these
found for EGFR, ErbB3, and ErbB4 (Fig. 2C). HCT116 cells
that stably express an empty vector or MUC1 were treated
with HRG. In concert with the findings in ZR-75-1 cells,
immunoblot analysis of anti-MUC1 immunoprecipitates with
anti-g-catenin demonstrated that HRG induces binding ofMUC1 and
g-catenin (Fig. 2D). These findings indicate thatHRG stimulates the
formation of MUC1-g-catenin complexes.
Nucleolar Localization of MUC1-c-Catenin ComplexesTo define the
subcellular localization of MUC1-g-catenin
complexes, ZR-75-1 cells were analyzed by confocal
microscopy after incubation with antibodies against MUC1
C-ter and g-catenin. The results show colocalization of
MUC1C-ter and g-catenin at the cell membrane (Fig. 3A). Bycontrast,
HRG stimulation for 20 min was associated with
localization of MUC1 C-ter in the nucleus (Fig. 3B). A
similar
pattern was observed for g-catenin, and overlay
demonstratedcolocalization with MUC1 C-ter (Fig. 3B). The
well-circum-
scribed colocalization of MUC1 and g-catenin in the
nucleussuggested a nucleolar pattern (Fig. 3B). Indeed, staining
with
an anti-nucleolin antibody confirmed HRG-induced redistri-
bution of MUC1 C-ter to the nucleolus (Fig. 3C). A similar
pattern of nucleolar colocalization for MUC1 C-ter with
g-catenin was observed in the ErbB2-positive MCF-7 breastcancer
cells (data not shown). Notably, stimulation of ZR-75-1
cells with EGF was associated with localization of MUC1
C-ter in a diffuse pattern throughout the nucleus (Fig. 3D).
Moreover, the lack of colocalization with nucleolin
indicated
that EGF induces nuclear targeting of MUC1 C-ter to
nonnucleolar sites (Fig. 3D). Following EGF stimulation,
nuclear MUC1 C-ter colocalizes with h-catenin and notg-catenin
(unpublished data).
Nucleolar Targeting of g-Catenin by MUC1766
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Role of MUC1 in the Subcellular Distribution of c-CateninTo
assess the functional role of MUC1 in g-catenin
signaling, HCT116/vector and HCT116/MUC1 cells were
analyzed for localization of g-catenin following HRG
stimu-lation. The confocal images show that g-catenin localizes to
thecell membrane of HCT116/vector cells (Fig. 4A). Moreover,
treatment of the HCT116/vector cells with HRG for 20 min had
no apparent effect on the distribution of g-catenin (Fig. 4A).In
HCT116/MUC1 cells, MUC1 C-ter and g-catenin werepredominantly
detectable at the cell membrane (Fig. 4B). By
contrast, HRG treatment of HCT116/MUC1 cells for 20 min
was associated with colocalization of MUC1 C-ter and g-cateninin
discrete nuclear structures (Fig. 4B). As found in ZR-75-1
cells, colocalization of MUC1 C-ter and nucleolin indicated
that MUC1 C-ter and g-catenin are targeted to the nucleolus(data
not shown).
Whereas a RRK motif in MUC1-CD may contribute to
nuclear localization, similar studies were performed on
HCT116 cells stably expressing a MUC1(RRK ! AAA)mutant.
Coimmunoprecipitation studies demonstrated that
binding of MUC1 to g-catenin is not affected by the RRK! AAA
mutation (data not shown). In contrast to HCT116/
FIGURE 1. HRG stimulates interaction of MUC1 and ErbB2. ZR-75-1
cells were left untreated or stimulated with 20-ng/ml HRG for 5
min. A. Lysateswere subjected to immunoprecipitation (IP ) with
anti-MUC1 (DF3) N-ter (left panel ) or anti-ErbB2 (right panel ).
Mouse IgG was used as a control. Theimmunoprecipitates were
analyzed by immunoblotting (IB ) with anti-ErbB2 and anti-MUC1
N-ter. Intensity of the signals was determined bydensitometric
scanning and compared with that obtained for untreated cells. B.
Lysates from control and HRG-treated ZR-75-1 cells were subjected
toimmunoprecipitation with anti-ErbB3 (left panel ) or anti-ErbB4
(right panel ). The immunoprecipitates were analyzed by
immunoblotting with the indicatedantibodies. ZR-75-1 cells were
grown to 60% confluence and incubated in medium with 0.1% serum for
24 h. The cells were left untreated (C),stimulated with 20-ng/ml
HRG for 5 min (D), or stimulated with 10-ng/ml EGF for 5 min (E),
fixed, and double stained with anti-MUC1 N-ter (green )
andanti-ErbB2 (red ).
Molecular Cancer Research 767
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
vector cells (Fig. 5A), MUC1 C-ter staining was intense over
the cell membrane of HCT116/MUC1(RRK ! AAA) cells(Fig. 5B).
Similar patterns were observed for g-catenin in bothHCT116/vector
and HCT116/MUC1(RRK ! AAA) cells (Fig.5, A and B). However, in
contrast to HCT116/vector cells
(Fig. 5A), stimulation of HCT116/MUC1(RRK ! AAA) cellswith HRG
for 20 min was associated with redistribution of
both MUC1 C-ter and g-catenin to the cytoplasm (Fig.
5B).Moreover, there was no detectable HRG-induced targeting of
MUC1 C-ter and g-catenin to the nucleolus (Fig. 5B).To extend
these observations, the localization of MUC1
C-ter and g-catenin was assessed by subcellular fractionationof
control and HRG-treated cells. Immunoblot analysis of the
nuclear fractions demonstrated that MUC1 C-ter is detectable
in the nuclei of HCT116/MUC1 cells but not of HCT116/
vector or HCT116/MUC1(RRK ! AAA) cells (Fig. 6). Theresults also
demonstrate that HRG increases nuclear targeting
of MUC1 C-ter in the HCT116/MUC1 cells (Fig. 6). More-
over, HRG treatment of HCT116/MUC1, but not HCT116/vector
or HCT116/MUC1(RRK ! AAA), was associated with anincrease in
nuclear g-catenin (Fig. 6). Equal loading of thenuclear fractions
was confirmed by immunoblotting for lamin B
(Fig. 6). Moreover, purity of the nuclear preparations was
demonstrated with antibodies against the cytosolic InBa,
themembrane-associatedMUC1N-ter subunit, and the endoplasmic
reticulum protein, calreticulin (Fig. 6). These findings
collec-
tively indicate that the RRK motif is important for
nucleolar
localization of MUC1 C-ter and g-catenin in the response toHRG
stimulation.
Confocal Microscopy of Human Breast CarcinomasTo define the
localization of MUC1 C-ter and g-catenin in
mammary tissues, confocal microscopy was first performed on
normal ductal epithelium. The results show localization of
MUC1 C-ter along the apical borders of the epithelial cells
lining the ducts (Fig. 7A). g-Catenin colocalized with MUC1C-ter
at the apical borders and was expressed at lateral borders
of the ductal epithelium (Fig. 7A). Little (if any) MUC1
C-ter
or g-catenin was detectable in the nucleus (Fig.
7A).Significantly, sections from ErbB2-positive breast
carcinomas
showed immunoflourescence staining of MUC1 C-ter and
g-catenin as discrete nuclear clusters (Fig. 7B). Sections
werealso stained with anti-MUC1 C-ter and antinucleolin. The
results demonstrate prominent colocalization of MUC1 C-ter
FIGURE 2. HRG stimulates the interaction between MUC1and
g-catenin. A. Lysates from ZR-75-1 cells left untreated
orstimulated with HRG for 5 min were subjected to
immuno-precipitation with anti-MUC1 N-ter or, as a control, IgG.
Theimmunoprecipitates were analyzed by immunoblotting withthe
indicated antibodies. B. Lysates from ZR-75-1 cells leftuntreated
or stimulated with 10-ng/ml EGF for 5 min weresubjected to
immunoprecipitation with anti-MUC1 or IgG. Theimmunoprecipitates
were analyzed by immunoblotting withthe indicated antibodies. C.
HCT116 cells were incubatedwith antibodies against the indicated
ErbB family members(open patterns ) or control mouse IgG (solid
patterns ) andanalyzed by flow cytometry. Similar results were
obtainedfor HCT116/MUC1 cells. D. HCT116/vector (HCT116/V )
andHCT116/MUC1 cells were left untreated or stimulated withHRG.
Anti-MUC1 N-ter immunoprecipitates were subjectedto immunoblotting
with anti-g-catenin or anti-MUC1 N-ter.
Nucleolar Targeting of g-Catenin by MUC1768
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
and nucleolin in breast carcinoma cells (Fig. 7C). Similar
results were obtained for g-catenin and nucleolin (Fig. 7D).
Theresults indicate that over 50% of the breast cancer cells
within
invasive islands exhibit nucleolar localization of MUC1
C-ter
and g-catenin. These findings in tissues and those in
culturedcells collectively demonstrate that MUC1-CD and g-catenin
aretargeted to nucleolus.
DiscussionInteraction of MUC1 and ErbB2
The MUC1 mucin-like glycoprotein is expressed on the
apical borders of normal mammary epithelium and at
substantially increased levels over the entire cell surface
of
breast carcinoma cells (15). Significantly, overexpression
of
MUC1 is associated with transformation as evidenced by
anchorage-independent growth and tumorigenicity (16). The
shed MUC1 N-ter is believed to function in the generation of
a
protective mucous barrier. The function of the C-ter, which
consists of an extracellular domain of f58 amino acids,
atransmembrane domain, and a 72-amino acid cytoplasmic tail,
is largely unknown. The finding that MUC1-CD binds directly
to h- and g-catenin suggested that the C-ter might function
intransducing signals from the cell surface to the interior of
the
cell (19). Indeed, the demonstration that MUC1-CD functions
as a substrate for GSK3h (20) and c-Src (21) has indicated
thatthe MUC1 C-ter may function in integrating signals from the
Wnt and growth factor receptor pathways. In this context,
activation of the EGFR is associated with tyrosine phospho-
rylation of MUC1-CD and regulation of the interaction
between
MUC1 and h-catenin (22, 27).Recent studies have shown that MUC1
associates with EGFR
and ErbB2–4 in pregnant and lactating mouse mammary glands
(27). The present work has explored the interaction between
MUC1 and ErbB2–4 in human breast cancer cells. The results
FIGURE 3. HRG induces nu-cleolar colocalization of MUC1C-ter and
g-catenin. ZR-75-1cells were grown to 60% conflu-ence and incubated
in mediumwith 0.1% serum for 24 h. Thecells were left untreated (A)
orstimulated with 20-ng/ml HRGfor 20 min (B), fixed, and
doublestained with anti-MUC1 C-ter(green ) and anti-g-catenin
(red).Nuclei were stained with SYN-TOX blue. High (�100)
(upperpanels ) and low (�63) (lowerpanels ) magnifications
areshown. ZR-75-1 cells were stim-ulated with 20-ng/ml HRG for20
min (C) or with 10-ng/mlEGF for 20 min (D), fixed, andstained with
anti-MUC1 C-ter andanti-nucleolin.
Molecular Cancer Research 769
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
of coimmunoprecipitation studies demonstrate the association
of
MUC1 with ErbB2–4. Significantly, treatment with HRG is
associated with increases in MUC1-ErbB2 complexes and
colocalization of these complexes in clusters at the cell
membrane (Fig. 8). Members of the ErbB family form both
homodimers and heterodimers in response to the diverse
ligands
that stimulate these receptors (1, 28). The available
evidence
suggests that ErbB2 functions as a coreceptor and is a
preferred
heterodimerization partner among the ErbB family members
(1, 28). In addition, ErbB2 is overexpressed in in situ and
invasive ductal carcinomas of the breast (28). The finding
that
HRG stimulates the association between ErbB2 and MUC1 may
therefore be of importance to ErbB2 signaling, particularly
in
tumors that overexpress both of these proteins.
Interaction of MUC1 and c-Cateninh- and g-catenin bind directly
to MUC1 at a SAGNGGSSL
motif in the CD (19). These vertebrate homologues of
Drosophila armadillo are found in the adherens junction
where they link E-cadherin to the actin cytoskeleton through
a-catenin (29). The finding that complexes between MUC1and h- or
g-catenin contain little (if any) a-catenin hassupported a function
distinct from their roles with E-cadherin
(19). In this regard, other studies have indicated that MUC1
and
E-cadherin compete for the same pool of h-catenin (20).Moreover,
negative regulation of the MUC1-h-catenin interac-tion by GSK3h is
associated with increased binding of h-catenin to E-cadherin (20).
In this model, down-regulation of
GSK3h by Wnt signaling would subvert E-cadherin functionin
homotypic cell-cell interactions by titrating binding of h-catenin
to MUC1. MUC1 is expressed along the apical borders
of normal ductal epithelial cells that are devoid of
cell-cell
interactions. By contrast, aberrant expression of MUC1 over
the entire surface of carcinoma cells may contribute to loss
of
E-cadherin function by disrupting interactions with h-
and/org-catenin.
The present results show that the MUC1-ErbB2 interaction
is associated with HRG-induced binding of MUC1 and g-catenin
(Fig. 8). HRG stimulation had less of an effect on
the interaction between MUC1 and h-catenin. Conversely,EGFR
signaling increases binding of MUC1 and h-catenin(22) but has
little effect on the interaction between MUC1 and
g-catenin. EGFR signaling also increases phosphorylation ofMUC1
on tyrosine in the SPY site (22), while HRG
stimulation had no apparent effect on tyrosine
phosphorylation
of MUC1-CD (data not shown). Activation of ErbB2, but not
FIGURE 4. MUC1 is neces-sary for HRG-induced targetingof MUC1
C-ter and g-catenin tothe nucleolus. HCT116/vector (A)and
HCT116/MUC1 (B) cells wereleft untreated or stimulated withHRG for
20 min. The cells wereassessed for reactivity with anti-MUC1 C-ter
and anti-g-catenin.Nuclei were stained with SYNTOXblue.
Nucleolar Targeting of g-Catenin by MUC1770
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
EGFR, in growth-arrested mammary acini results in
reinitiation
of proliferation, disruption of tight junctions, loss of
polarity,
and filled lumina (11). These results indicate that ErbB2
activation can selectively disrupt regulation of mammary
epi-
thelial cell proliferation and organization. Other effectors,
such
as Rac, Cdc42, and PI3K, which induce invasiveness of
mammary epithelial cells, may cooperate with ErbB2 in
disrupting polarized epithelia (30). One report has also
indicated that ErbB2 suppresses E-cadherin expression in
mammary epithelial cells (31), but such regulation was not
found in other studies (11). The present findings provide
evidence for the involvement of ErbB2 activation and the
regulation of g-catenin signaling as another potential
mecha-nism for increasing invasiveness. Thus, HRG-induced
increases
in binding of g-catenin to MUC1 could decrease the
availabilityof g-catenin for linking E-cadherin to the actin
cytoskeletonand thereby disrupt homotypic cell-cell signaling.
Nucleolar Localization of MUC1 C-Ter and c-CateninThe present
results further indicate that HRG stimulation
is associated with nuclear targeting of MUC1 C-ter and g-catenin
(Fig. 8). The well-circumscribed nuclear distribution
of the MUC1 C-ter signal and colocalization with anti-
nucleolin staining supported compartmentalization of MUC1
C-ter in the nucleolus. Similar results were obtained with
g-catenin, supporting the likelihood that the MUC1-g-catenincomplex
is targeted to the nucleolus in response to HRG
stimulation. In concert with these findings, MUC1 C-ter and
g-catenin are detectable in nucleoli of ErbB2-positive
primarybreast carcinomas. The observation that over 50% of the
breast cancer cells exhibit nucleolar colocalization of MUC1
C-ter and g-catenin indicate that, as found in vitro , MUC1may
interact with the ErbB2 signaling pathway in primary
breast carcinomas. The nucleolus is a membrane-free nuclear
subdomain in which rRNAs are transcribed and processed
into ribosome subunits (32). Additional functions that may
be
attributable to the nucleolus include the processing of
other
ribonucleoproteins (33, 34) and export of mRNAs and
tRNAs (35, 36). In addition, the nucleolus may function in
sequestering specific regulatory factors (37). For example,
Mdm2 is sequestered in the nucleolus by an ARF-dependent
mechanism (38–40). Disassembly of the nucleolus during
cell cycle progression can in turn release sequestered
factors.
In the nucleus, g-catenin interacts with the T-cell
factor/lymphoid enhancer factor transcription factors and
functions
as a coactivator. Like h-catenin, g-catenin can contribute
tocell transformation by a mechanism involving transactivation
of
c-Myc expression (41).
FIGURE 5. Nucleolar locali-zation of MUC1 C-ter and g-catenin is
conferred by theMUC1 RRK motif. HCT116/vec-tor (A) and
HCT116/MUC1(RRK! AAA) (B) cells were left un-treated or stimulated
with HRGfor 20 min. Cells were analyzedfor staining with anti-MUC1
C-terand anti-g-catenin. Morphology ofthe cells was visualized by
bright-field microscopy.
Molecular Cancer Research 771
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Activation of the Wnt signaling pathway is associated with
accumulation of h- and g-catenin in the nucleus. Themechanisms
responsible for targeting h- and g-catenin to thenucleus are not
clear. Neither protein has a definitive nuclear
localization signal; however, h-catenin is imported into
thenucleus by binding directly to the nuclear pore machinery
(42). Moreover, binding to T-cell factor/lymphoid enhancer
factor transcription factors is probably not responsible for
nuclear localization of h-catenin (43). The adenomatouspolyposis
coli protein can function as a h-catenin chaperonein nuclear export
but apparently not in nuclear import (44, 45).
Recent studies have demonstrated that MUC1 colocalizes with
h-catenin in the nucleus and increases nuclear levels of h-
catenin (16, 24). These findings have indicated that MUC1
may function in the import and/or stabilization of nuclear
h-catenin. Importantly, the nuclear colocalization of
MUC1-h-catenin complexes is found outside the nucleolus (16, 24,and
unpublished data).
The present results in HCT116/vector and HCT116/MUC1
cells indicate that HRG-induced nucleolar localization of
g-catenin is dependent on MUC1 expression. The MUC1-CDcontains a
RRK motif that may function as a monopartite
nuclear localization signal (46). Studies of the c-Myc
nuclear
localization signal (PAAKRVKLD) have demonstrated the
functional role of neutral amino acids and the dipeptide LD
in
nuclear targeting (47). The RRK basic cluster in the MUC1-CD
is also flanked by neutral amino acids and the LD dipeptide
(CQCRRKNYGQLD). Importantly, mutation of the MUC1
RRK motif to AAA abrogated HRG-induced nucleolar
localization of MUC1 C-ter. In addition, targeting of
g-cateninto the nucleolus in response to HRG was not found in
cells
expressing the MUC1(RRK ! AAA) mutant. These findingsprovide the
first evidence that MUC1 functions in nuclear
signaling and that g-catenin is transported to the nucleolus by
aMUC1-dependent mechanism.
Materials and MethodsCell Culture
Human ZR-75-1 and MCF-7 breast carcinoma cells
(American Type Culture Collection, Manassas, VA) were
cultured in RPMI 1640 high-glucose medium containing 10%
heat-inactivated fetal bovine serum (HI-FBS), 100-U/ml
penicillin, 100-Ag/ml streptomycin, and 2-mM L-glutamine.HCT116
colon carcinoma cells (American Type Culture
Collection) were grown in DMEM containing 10% HI-FBS
and antibiotics. Cells were maintained in medium with 0.1%
HI-FBS for 24 h and stimulated with 20-ng/ml HRG or 10-ng/
ml EGF (Calbiochem-Novabiochem, San Diego, CA) at 37jC.
Cell TransfectionspIRESpuro2, pIRESpuro2-MUC1, and
pIRESpuro2-
MUC1(RRK ! AAA) were transfected into HCT116 cells
byLipofectAMINE. Stable transfectants were selected in the
presence of 0.4-Ag/ml puromycin (Calbiochem-Novabiochem).
Immunoprecipitation and ImmunoblottingLysates were prepared from
subconfluent cells as described
(20). Equal amounts of cell lysate protein were incubated
with
antibody DF3 (anti-MUC1) (15), anti-ErbB2 (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-ErbB3 (Santa Cruz
Biotechnology), anti-ErbB4 (Santa Cruz Biotechnology), or
mouse IgG. The immune complexes were prepared as described
(20), separated by SDS-PAGE, and transferred to
nitrocellulose
membranes. The immunoblots were probed with anti-MUC1,
anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-h-catenin (Zymed,
SanFrancisco, CA), or anti-g-catenin (Zymed). Reactivity
wasdetected with horseradish peroxidase-conjugated second anti-
bodies and chemiluminescence (Perkin-Elmer Corp., Boston,
MA).
Immunoflourescence Confocal MicroscopyCultured cells were washed
three times in PBS (containing
FIGURE 6. HRG-induced nuclear localization of MUC1 and
g-catenin.Nuclear fractions were analyzed by immunoblotting with
the indicatedantibodies. Whole cell lysates (WCL ) were used as a
positive control.
Nucleolar Targeting of g-Catenin by MUC1772
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Mg2+ and Ca2+), fixed with 3.7% formaldehyde in buffer A
(PBS containing 10-AM ZnCl2) for 10 min, permeabilized with0.25%
Triton X-100/3.7% formaldehyde in buffer A for 5 min,
and postfixed with 3.7% formaldehyde in buffer A for 5 min.
The cells were then washed three times with PBS and
incubated with blocking buffer (PBS containing 4%
protease-free BSA and 5% normal goat serum). Incubation
with anti-MUC1, anti-ErbB2, anti-MUC1 C-ter (Neomarkers,
FIGURE 7. Colocalization of MUC1C-ter and g-catenin to the
nucleolus ofhuman breast carcinoma cells. Sectionsof normal mammary
ductal epithelium (A)and two anti-HER2/ErbB2-positive pri-mary
invasive ductal breast carcinomas(B, upper and lower panels ) were
as-sessed for reactivity with anti-MUC1 C-terand anti-g-catenin.
Morphology was visu-alized at high and low (inset ) power byH&E
staining. Breast carcinoma cells werestained with anti-MUC1 C-ter
(C) or anti-g-catenin (D) and anti-nucleolin.
Molecular Cancer Research 773
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Fremont, CA), anti-g-catenin, and anti-nucleolin
(ResearchDiagnostics, Flanders, NJ) in blocking buffer was
performed
overnight at 4jC. The cells were washed with PBS,
incubatedovernight with secondary FITC- or Texas Red-conjugated
goat
anti-hamster or anti-mouse IgG antibodies (Jackson Immuno-
Research Laboratories, West Grove, PA) at 4jC, washed withPBS,
washed three times with buffer B (20-mM Tris, pH 7.5,
0.15-M NaCl), and stained with 0.2-AM of SYNTOX BlueNuclei C
solution for 2 h. After washing again with buffer B, the
cells were mounted with Slowfade solution and analyzed by
confocal microscopy using an inverted Zeiss LSM510 scope
(Carl Zeiss, Inc., Thornwood, NY). Images were captured at
0.6-nm increments along the Z axis and converted to
composites
by LSM510 software version 3.0.
Flow CytometryCells were incubated with anti-EGFR, anti-ErbB2,
anti-
ErbB3, anti-ErbB4, or mouse IgG for 30 min, washed,
incubated with goat antimouse immunoglobulin-flourescein-
conjugated antibody (Santa Cruz Biotechnology), and fixed
in 1% formaldehyde/PBS. Reactivity was detected by immu-
noflourescence FACScan.
Subcellular FractionationPreparation of nuclear fractions was
performed as described
(48). Purity of the fractionations was monitored by
immunoblot
analysis with anti-lamin B (Oncogene Science, Cambridge,
MA), anti-calreticulin (Santa Cruz Biotechnology) and anti-
InBa (Santa Cruz Biotechnology) antibodies.
AcknowledgmentsThe authors acknowledge Kamal Chauhan for
excellent technical support. D.K.has a financial interest in
ILEX.
References1. Olayioye, M. A., Neve, R. M., Lane, H. A., and
Hynes, N. E. The ErbBsignaling network: receptor heterodimerization
in development and cancer.EMBO J., 19: 3159–3167, 2000.
2. Carraway, K. L., III and Cantley, L. C. A neu acquaintance
for erbB3 anderbB4: a role for receptor heterodimerization in
growth signaling. Cell, 78: 5– 8,1994.
3. Riese D. J., II and Stern, D. F. Specificity within the EGF
family/ErbBreceptor family signaling network. Bioessays, 20: 41–48,
1998.
4. Wada, T., Qian, X. L., and Greene, M. I. Intermolecular
association of thep185neu protein and EGF receptor modulates EGF
receptor function. Cell, 61:1339– 1347, 1990.
5. Plowman, G. D., Culouscou, J. M., Whitney, G. S., Green, J.
M., Carlton,G. W., Foy, L., Neubauer, M. G., and Shoyab, M.
Ligand-specific activation ofHER4/p180erbB4, a fourth member of the
epidermal growth factor receptorfamily. Proc. Natl. Acad. Sci. USA,
90: 1746– 1750, 1993.
6. Tzahar, E., Waterman, H., Chen, X., Levkowitz, G.,
Karunagaran, D., Lavi, S.,Ratzkin, B. J., and Yarden, Y. A
hierarchical network of interreceptor interactionsdetermines signal
transduction by Neu differentiation factor/neuregulin andepidermal
growth factor. Mol. Cell. Biol., 16: 5276–5287, 1996.
7. Graus-Porta, D., Beerli, R. R., Daly, J. M., and Hynes, N. E.
ErbB-2, thepreferred heterodimerization partner of all ErbB
receptors, is a mediator of lateralsignaling. EMBO J., 16:
1647–1655, 1997.
8. Olayioye, M. A., Graus-Porta, D., Beerli, R. R., Rohrer, J.,
Gay, B., andHynes, N. E. ErbB-1 and ErbB-2 acquire distinct
signaling properties dependentupon their dimerization partner. Mol.
Cell. Biol., 18: 5042– 5051, 1998.
9. Ricci, A., Lanfrancone, L., Chiari, R., Belardo, G., Pertica,
C., Natali, P. G.,Pelicci, P. G., and Segatto, O. Analysis of
protein-protein interactions involved inthe activation of the
Shc/Grb-2 pathway by the ErbB-2 kinase. Oncogene, 11:1519– 1529,
1995.
10. Zrihan-Licht, S., Deng, B., Yarden, Y., McShan, G., Keydar,
I., and Avraham,H. Csk homologous kinase, a novel signaling
molecule, directly associates withthe activated ErbB-2 receptor in
breast cancer cells and inhibits their proliferation.J. Biol.
Chem., 273: 4065–4072, 1998.
11. Muthuswamy, S. K., Gilman, M., and Brugge, J. S. Controlled
dimerizationof ErbB receptors provides evidence for differential
signaling by homo- andheterodimers. Mol. Cell. Biol., 19:
6845–6857, 1999.
12. Janda, E., Litos, G., Grunert, S., Downward, J., and Beug,
H. OncogenicRas/Her-2 mediate hyperproliferation of polarized
epithelial cells in 3Dcultures and rapid tumor growth via the PI3K
pathway. Oncogene, 21: 5148–5159, 2002.
13. Polakis, P. Wnt signaling and cancer. Genes Dev., 14:
1837–1851, 2000.
14. Lin, S. Y., Xia, W., Wang, J. C., Kwong, K. Y., Spohn, B.,
Wen, Y., Pestell,R. G., and Hung, M. C. h-catenin, a novel
prognostic marker for breast cancer: itsroles in cyclin D1
expression and cancer progression. Proc. Natl. Acad. Sci. USA,97:
4262–4266, 2000.
15. Kufe, D., Inghirami, G., Abe, M., Hayes, D., Justi-Wheeler,
H., andSchlom, J. Differential reactivity of a novel monoclonal
antibody (DF3)with human malignant versus benign breast tumors.
Hybridoma, 3: 223 –232,1984.
16. Li, Y., Liu, D., Chen, D., Kharbanda, S., and Kufe, D. Human
DF3/MUC1carcinoma-associated protein functions as an oncogene.
Oncogene, 22: 6107–6110, 2003.
17. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard,
J., and Burchell,J. A. A highly immunogenic region of a human
polymorphic epithelial mucinexpressed by carcinomas is made up of
tandem repeats. J. Biol. Chem., 263:12820 –12823, 1988.
18. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and
Kufe, D.Isolation and sequencing of a cDNA coding for the human DF3
breastcarcinoma-associated antigen. Proc. Natl. Acad. Sci. USA, 85:
2320– 2323,1988.
19. Yamamoto, M., Bharti, A., Li, Y., and Kufe, D. Interaction
of the DF3/MUC1breast carcinoma-associated antigen and h-catenin in
cell adhesion. J. Biol.Chem., 272: 12492 –12494, 1997.
20. Li, Y., Bharti, A., Chen, D., Gong, J., and Kufe, D.
Interaction of glycogensynthase kinase 3h with the DF3/MUC1
carcinoma-associated antigen andh-catenin. Mol. Cell. Biol., 18:
7216–7224, 1998.
21. Li, Y., Kuwahara, H., Ren, J., Wen, G., and Kufe, D. The
c-Src tyrosinekinase regulates signaling of the human DF3/MUC1
carcinoma-associatedantigen with GSK3h and h-catenin. J. Biol.
Chem., 276: 6061–6064, 2001.
22. Li, Y., Ren, J., Yu, W.-H., Li, G., Kuwahara, H., Yin, L.,
Carraway, K. L.,and Kufe, D. The EGF receptor regulates interaction
of the human DF3/MUC1
FIGURE 8. Schematic representation of the involvement of MUC1
inHRG-induced targeting of g-catenin to the nucleolus.
Nucleolar Targeting of g-Catenin by MUC1774
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
carcinoma antigen with c-Src and h-catenin. J. Biol. Chem., 276:
35239–35242, 2001.
23. Takeichi, M. Cadherins: a molecular family important in
selective cell-celladhesion. Annu. Rev. Biochem., 59: 237– 252,
1990.
24. Li, Y., Chen, W., Ren, J., Yu, W., Li, Q., Yoshida, K., and
Kufe, D. DF3/MUC1 signaling in multiple myeloma cells is regulated
by interleukin-7. CancerBiol. Ther., 2: 187–193, 2003.
25. Shimizu, H., Koyama, N., Asada, M., and Yoshimatsu, K.
Aberrantexpression of integrin and erbB subunits in breast cancer
cell lines. Int. J.Oncol., 21: 1073–1079, 2002.
26. Ren, J., Li, Y., and Kufe, D. Protein kinase C y regulates
function of the DF3/MUC1 carcinoma antigen in h-catenin signaling.
J. Biol. Chem., 277: 17616–17622, 2002.
27. Schroeder, J., Thompson, M., Gardner, M., and Gendler, S.
TransgenicMUC1 interacts with epidermal growth factor receptor and
correlates withmitogen-activated protein kinase activation in the
mouse mammary gland. J. Biol.Chem., 276: 13057 –13064, 2001.
28. Harari, D. and Yarden, Y. Molecular mechanisms underlying
ErbB2/HER2action in breast cancer. Oncogene, 19: 6102–6114,
2000.
29. Hulsken, J., Birchmeier, W., and Behrens, J. E-cadherin and
APC competefor the interaction with h-catenin and the cytoskeleton.
J. Cell Biol., 127: 2061–2069, 1994.
30. Keely, P. J., Westwick, J. K., Whitehead, I. P., Der, C. J.,
and Parise, L. V.Cdc42 and Rac1 induce integrin-mediated cell
motility and invasiveness throughPI(3)K. Nature, 390: 632– 636,
1997.
31. D’Souza, B. and Taylor-Papadimitriou, J. Overexpression of
ERBB2in human mammary epithelial cells signals inhibition of
transcription of theE-cadherin gene. Proc. Natl. Acad. Sci. USA,
91: 7202–7206, 1994.
32. Shaw, P. J. and Jordan, E. G. The nucleolus. Annu. Rev. Cell
Dev. Biol., 11:93–121, 1995.
33. Politz, J. C., Yarovoi, S., Kilroy, S. M., Gowda, K., Zwieb,
C., and Pederson,T. Signal recognition particle components in the
nucleolus. Proc. Natl. Acad. Sci.USA, 97: 55–60, 2000.
34. Lange, T. S. and Gerbi, S. A. Transient nucleolar
localization of U6 smallnuclear RNA in Xenopus laevis oocytes. Mol.
Biol. Cell, 11: 2419–2428, 2000.
35. Schneiter, R., Kadowaki, T., and Tartakoff, A. M. mRNA
transport in yeast:time to reinvestigate the functions of the
nucleolus. Mol. Biol. Cell, 6: 357 –370,1995.
36. Bertrand, E., Houser-Scott, F., Kendall, A., Singer, R. H.,
and Engelke, D. R.Nucleolar localization of early tRNA processing.
Genes Dev., 12: 2463–2468,1998.
37. Visintin, R. and Amon, A. The nucleolus: the magician’s hat
for cell cycletricks. Curr. Opin. Cell Biol., 12: 372 –377,
2000.
38. Zhang, Y. and Xiong, Y. Mutations in human ARF exon 2
disrupt itsnucleolar localization and impair its ability to block
nuclear export of MDM2 andp53. Mol. Cell, 3: 579– 591, 1999.
39. Tao, W. and Levine, A. p19ARF stabilizes p53 by blocking
nucleo-cytoplasmic shuttling of mdm2. Proc. Natl. Acad. Sci. USA,
96: 6937–6941,1999.
40. Lohrum, M. A., Ashcroft, M., Kubbutat, M. H., and Vousden,
K. H.Identification of a cryptic nucleolar-localization signal in
MDM2. Nat. Cell Biol.,2: 179– 181, 2000.
41. Kolligs, F. T., Kolligs, B., Hajra, K. M., Hu, G., Tani, M.,
Cho, K. R.,and Fearon, E. R. g-catenin is regulated by the APC
tumor suppressor andits oncogenic activity is distinct from that of
h-catenin. Genes Dev., 14:1319– 1331, 2000.
42. Fagotto, F., Gluck, U., and Gumbiner, B. M. Nuclear
localization signal-independent and
importin/karyopherin-independent nuclear import of h-catenin.Curr.
Biol., 8: 181–190, 1998.
43. Prieve, M. G. and Waterman, M. L. Nuclear localization and
formation ofh-catenin-lymphoid enhancer factor 1 complexes are not
sufficient for activationof gene expression. Mol. Cell. Biol., 19:
4503–4515, 1999.
44. Henderson, B. R. Nuclear-cytoplasmic shuttling of APC
regulates h-cateninsubcellular localization and turnover. Nat. Cell
Biol., 2: 653 –660, 2000.
45. Neufeld, K. L., Zhang, F., Cullen, B. R., and White, R. L.
APC-mediateddownregulation of h-catenin activity involves nuclear
sequestration and nuclearexport. EMBO Rep., 1: 519 –523, 2000.
46. Dingwell, C. and Laskey, R. A. Nuclear targeting sequences—a
consensus?Trends Biochem. Sci., 16: 478–482, 1991.
47. Makkerh, J. P., Dingwall, C., and Laskey, R. A. Comparative
mutagenesis ofnuclear localization signals reveals the importance
of neutral and acidic aminoacids. Curr. Biol., 6: 1025–1027,
1996.
48. Kharbanda, S., Saleem, A., Yuan, Z.-M., Kraeft, S.,
Weichselbaum, R., Chen,L. B., and Kufe, D. Nuclear signaling
induced by ionizing radiation involvescolocalization of the
activated p56/p53lyn tyrosine kinase with p34cdc2. CancerRes., 56:
3617–3621, 1996.
Molecular Cancer Research 775
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
2003;1:765-775. Mol Cancer Res Yongqing Li, Wei-hsuan Yu, Jian
Ren, et al. W.-h.Y. contributed equally to this work.National
Cancer Institute grant CA97098. Note: Y.L. and
1 1Mechanism Dependent on the DF3/MUC1 Oncoprotein-Catenin to
the Nucleolus by aγHeregulin Targets
Updated version
http://mcr.aacrjournals.org/content/1/10/765
Access the most recent version of this article at:
Cited articles
http://mcr.aacrjournals.org/content/1/10/765.full#ref-list-1
This article cites 48 articles, 28 of which you can access for
free at:
Citing articles
http://mcr.aacrjournals.org/content/1/10/765.full#related-urls
This article has been cited by 21 HighWire-hosted articles.
Access the articles at:
E-mail alerts related to this article or journal.Sign up to
receive free email-alerts
Subscriptions
Reprints and
[email protected] at
To order reprints of this article or to subscribe to the
journal, contact the AACR Publications
Permissions
Rightslink site. (CCC)Click on "Request Permissions" which will
take you to the Copyright Clearance Center's
.http://mcr.aacrjournals.org/content/1/10/765To request
permission to re-use all or part of this article, use this link
on April 5, 2021. © 2003 American Association for Cancer
Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/content/1/10/765http://mcr.aacrjournals.org/content/1/10/765.full#ref-list-1http://mcr.aacrjournals.org/content/1/10/765.full#related-urlshttp://mcr.aacrjournals.org/cgi/alertsmailto:[email protected]://mcr.aacrjournals.org/content/1/10/765http://mcr.aacrjournals.org/